lysis buffer----SDS or PMSF? - (Aug/10/2005 )

I have two recipes in my hand for RIPA buffer.

1. 50mM Tris-Hcl, PH7.4; NP-40 1%, Na-DOC 0.25%; Nacl 150mM; EDTA 1mM; PMSF 1mM; Protease cocktail 1Tab; Na3VO4 1mM; NaF 1mM;

2. No PMSF in above recipe, but added 0.1% SDS instead.

I am using the 2nd recipe now to lysate the spinal cord tissue, and after homogenizing the tissue, I put them at 37 degree celcieus and then centrifuge at 14000rpm for 10 min. Collect the supernatent for membrane protein electrophoresis. etc.

My question is :
1. Do I have to add PMSF in my lysis buffer since it is regularly used in other recipes? or SDS is not compatible with PMSF, so I should select either of them?

2.Is it reasonable to put the lysate at 37 degree celcieus before centrifuging?

3. I usually boil the sample at 95 degree for 5 minutes. I was told the other day that membrane protein is not eligible for boliling , he suggested 37 degree for 30min before loading. I have prepared the same sample under two conditions and ran them in the same gel, the latter gives much denser band. Anybody had similar experience?

4. My western blot went well with SDS-PAGE gel; but when switched to Nu-PAGE Bis-Tris gel, I always get smeared band (120Kd) after transfer, though actin band looks perfect. my sampel was denatured at 70 degree for 10min before loading. This had anything to do with SDS in my lysis buffer or putting the lysate at 37 degree celcieus before centrifuging?

Too many questions, huh? When you are not getting the result you expect to see, you just have to worry about if you did anything wrong!

1
sds and PMSF are compatibles. You have cocktail in your buffer that can replace PMSF. so no strict need to add PMSF.

2
if yourprotein sample is good, why not? But i do the lysis in ice.

3
...

4
sorry nothing to tell.

enjoy your exps.
Fred